Pharmaceutically active conjugates having improved body tissue binding specificity

ABSTRACT

Pharmaceutically active conjugates comprising a pharmaceutically active substance for treating a disorder of the body that involves a specified body tissue conjugated directly or indirectly with at least one fragment of an adhesive glycoprotein such as fibronectin, the said glycoprotein fragment(s) having improved binding specificity compared with the parent protein for the said body tissue.

This invention relates to pharmaceutically active conjugates havingimproved binding specificity for specified body tissue. The inventionfurther relates to methods of preparing these conjugates and topharmaceutical preparations that contain them. In particularembodiments, the invention relates to pharmaceutically-active conjugatescontaining either plasminogen activators or anti-rheumatic drugs.

It is common practice to administer large doses of various drugs to thebody to treat conditions which may only affect a small region of thebody. Large doses are often required in order to attain a sufficientconcentration of the drug at the target area. However, these large andconstantly-administered doses can produce serious side effects inpatients being treated, which has often lead to treatment beingsuspended even though improvement in the treated condition might betaking place. This problem has existed in the past particularly in theadministration of toxic anti-tumour drugs for destroying cancer cellsand in the administration of toxic anti-rheumatic drugs, especially goldcompounds.

One further example of the administration of drugs where this problemexists is in the treatment of thrombotic conditions. Conventionaltreatment for certain thrombotic conditions, such as deep veinthrombosis or pulmonary thrombosis, involves continuous infusion ofagents which stimulate fibrinolysis Fibrinolysis is the term given tothe process of proteolytic degradation of a fibrin-based clot.Thrombolysis is the plasmin mediated proteolysis which brings about thebreak up of large vascular obstructions. It is generally accepted thatthe main enzyme responsible for fibrinolysis is plasminogen-plasmin. Thezymogen, plasminogen, is converted by one of a range of activators toplasmin, which is an active protease capable of degrading a cross-linkedfibrin clot to soluble products (FDPs). Plasmin is a serine protease,produced from plasminogen by limited proteolytic cleavage accompanied bya conformational change.

The precise mechanism of plasminogen activation depends on theplasminogen activator involved. Thus, activation can also occur withoutproteolytic cleavage in the case of plasminogen activators from certainmicroorganisms which act allosterically, e.g. streptokinase. In generalactivation is more effective if it occurs on the surface of the clot, aphenomenon aided by the affinity that plasminogen has for fibrin.Plasminogen activators occur in blood, a variety of tissues and in bodyfluids such as urine, saliva and semen. As hereinbefore indicated, theyare also produced by certain microorganisms. Small quantities of alabile plasminogen activator, tissue plasminogen activator (t-PA), occurin the circulation. Its level is raised following stimuli includingexercise and venous occlusion. t-PA, which carries a fibrin-bindingregion, has been isolated from cadaveric plasma and from culture mediumof vascular endothelial cells and the Bowes melanoma cell line. Morerecently, this plasminogen activator has also been prepared byrecombinant DNA technology (see, for example, GB-A No. 2119804).

Urokinase (UK) is a plasminogen activator present in urine. Unlike t-PA,it can be readily isolated in a highly purified, crystalline form. It isa single chain β-globulin which exists in two forms of molecular weights54,000 and 32,000 respectively. UK is (apparently) synthesised in thekidney and, unlike t-PA, it has little affinity for fibrin. UK activatesplasminogen to produce plasmin which is subject to inhibition.

Streptokinase (SK) is a plasminogen activator produced by β-haemolyticstreotococci and available in purified preparations. SK is a singlechain α2-globulin having a molecular weight around 46,000. It acts as anactivator by binding to plasminogen causing a conformational change. Theresultant SK-plasminogen complex possesses enzymic activity, but is notinhibited by α2-macroglobulin.

Both UK and SK have been used successfully as thrombolytic agents. SK ismore commonly used since it is cheaper to produce, although UK isgenerally regarded as a better agent. SK is highly antigenic and tendsto be effective for only one treatment, after which the number ofantibodies raised by the body's autoimmune system render subsequenttreatments much less effective. In addition, the titre of anti-SKantibodies varies between individuals depending on the previous historyof Streptococcal infections. This makes it difficult to determine theeffective safe dose. Antibody mediated resistance does not occur withUK.

Although UK has no antigenicity, UK therapy suffers from the dualdisadvantages of limited availability and high cost, consequent on theneed to fractionate very large volumes of human urine. The huge dose ofUK necessary to maintain a thrombolytic state is thought to be partlydue to its poor affinity for fibrin. The result is that a completecourse of treatment requires the fractionation of around 5000 liters ofurine. In the case of both UK and SK therapy, there is a serious risk ofhaemorrhagic complication due to the systemic administration of largedoses of activator. Attempts to achieve thrombolysis by localadministration have not been encouraging. In the case of SK, it isthought that effective and haemorrhagic doses are almost the same, sothat an effective dose can cause hyperplasminaemia, fibrinogendegradation, and an accumulation of fibrinogen degradation productswhich have an anticoagulant effect and so add to haemorrhagiccomplications.

More recently, attempts have been made to overcome this problem bylinking drugs to carriers which have a high affinity for the area of thebody requiring treatment. These carriers target the drug on to the arearequiring treatment and can thereby be administered in relatively lowbut still effective doses. Examples of such carriers are disclosed inpublished patent specifications EP-A No. 2-114685 and, more recently,U.S. Pat. No. 4,587,122, which describe the preparation and use ofvarious anti-tumour, anti-bacterial and anti-inflammatory drugscovalently conjugated with whole fibronectin (an adhesive glycoprotein)using protein cross-linking agents. Although fibronectin is claimed tobe an effective carrier which binds readily to morbid regions of thebody where treatment by drugs might be required, fibronectin itselfcarries binding sites for a large number of body tissues and sopreferential binding to a site of the body requiring treatment cannot beguaranteed; indeed, preferential binding may occur in other parts of thebody in which case the amount of drug accumulating in the area requiringtreatment may in some cases be negligible. Furthermore, there is someevidence to suggest that fibronectin normally accumulates at siteswithin the body by self-association on to initial, bound fibronectin.That is to say, self-association leads to non-specific amplification offibronectin accumulation. Clearly, this mechanism would severely impairthe "targetting" ability of administered fibronectin-drug conjugatessince they could bind to any site of fibronectin accumulation within thebody.

The present invention is based on the concept of providing improved drugconjugates having targetting portions which have a higher relativedegree of binding specificity for particular areas of the body wheredrug treatment is required and which have a reduced tendency toself-associate. The present invention thus seeks to provide a solutionto the problem of how to fulfill this need by providing targettingportions of pharmaceutically-active conjugates in the form of proteinfragments, e.g. generated by the enzymic digestion of adhesiveglycoproteins, which have affinity for a specified body tissue involvedin a bodily disorder. One advantage of employing these fragments is thatit is possible to prepare adhesive glycoprotein fragments which havespecificity or at least a high degree of selectivity for single bodytissue types, whereas whole adhesive glycoproteins, such as intactfibronectin, usually have a broad range affinity for a number of bodytissues. The use of protein fragments therefore makes it possible toprepare conjugates that will bind substantially only to the specifiedbody tissue involved in the disorder being treated. Furthermore, it hasbeen found using fibronectin as an appropriate model, that proteinfragments have a reduced tendency to bind under physiological conditionsto their parent glycoprotein, which enhances their specificity ofbinding within the whole body.

According to a first aspect of the present invention, therefore, thereis provided a pharmaceutically active conjugate comprising apharmaceutically active substance for treating a disorder of the bodythat involves a specified body tissue characterised in that saidpharmaceutically active substance is conjugated directly or indirectlywith at least one fragment of an adhesive glycoprotein having improvedbinding specificity compared with the parent protein for the said bodytissue.

The glycoprotein will normally possess binding sites specific to atleast two different tissues.

Protein fragments suitable for a conjugate according to the presentinvention may be derived for example by protease digestion of anaturally-occurring adhesive glycoprotein or portion thereof, inglycosylated or non-glycosylated form, or from a genetically engineeredequivalent thereof having the same amino acid sequence or the same aminoacid sequence apart from one or more changes which do not affect bindingspecificity. Moreover, it will be appreciated that suitable proteinfragments for a conjugate of the present invention may alternatively beprepared directly by recombinant DNA technology or chemical synthesis.The pharmaceutically active substance will preferably be covalentlyconjugated to the chosen protein fragment or fragments directly orindirectly through a cross-linking reagent or via a carrier moleculewhich may carry a number of molecules of an active substance as well asone or more fragments of an adhesive protein.

A pharmaceutically active conjugate of this type can be administered bynormal means (usually orally or intraveneously), and yet is more readilyand efficiently targetted on to particular sites of the body which areinvolved in a specified bodily disorder. Such a conjugate will tend tobind with a high degree of selectivity to a site of interest readily andrapidly as it is carried past the site within the blood stream oranother body fluid and so the tendency for competing side reactions tooccur between the active substance in the conjugate and other bodytissues will be reduced. This has the dual advantage of decreasing thedosage requirements for treating a specified disorder whilst at the sametime reducing the extent of undesirable side effects that are frequentlyassociated with the administration of large quantities ofinefficiently-utilised pharmaceutically active substances to the body.

Adhesive glycoproteins and certain digestion fragments thereof are knownto have a strong affinity for certain proteins produced by the body.Preferred conjugates of the present invention are therefore of a typethat are effective for treating disorders of the body involving anaccumulation of tissue containing or consisting of protein matter. Inthis case, the pharmaceutically active substance may preferably comprisean agent for bringing about the proteolytic degradation of theaccumulated protein. Where the accumulated protein consists of afibrin-based clot, a suitable conjugate of the present invention willcomprise a plasminogen activator, for example t-PA, streptokinase (SK)or urokinase (UK), conjugated to a protein fragment with a high degreeof selectivity for binding to fibrin, preferably a fibronectin fragmentwith binding affinity for fibrin, but which unlike whole fibronectinsubstantially lacks gelatin or collagen binding affinity. Indeed,conjugates of the present invention have been found to be particularlyeffective when the pharmaceutically active substance is in the form of aprotein or enzyme because these can be readily conjugated throughcovalent bonding to glycoprotein fragments.

Further examples of preferred conjugates according to the presentinvention are anti-rheumatic drug-protein conjugates suitable forselectively targetting the chosen drug to articular tissue in jointsaffected by rheumatoid arthritis. This condition is characterised bychronic synovial inflammation leading to release and activation ofcollagenase with the result that collagen in the connective tissue ofaffected joints is broken down and denatured to form gelatin. Thus, aconjugate of the present invention for treatment of rheumatoid arthritiswill comprise an anti-rheumatic drug such as gold, a gold compound orpenicillamine, bound directly or indirectly to one or more proteinfragments with a high degree of selectivity for gelatin-binding in thebody. As hereinbefore indicated, fibronectin has a gelatin-bindingdomain and moreover has a higher affinity for gelatin (denaturedcollagen) than native collagen. Consequently, it is most preferred toemploy as the protein fragment component of an anti-rheumatic drugconjugate of the present invention one or more fibronectin fragmentscomprising a gelatin-binding domain, but having substantially nofibrin-binding affinity.

Other pharmaceutically active substances which may be conjugated withprotein fragments to form a conjugate of the present invention areanti-tumour agents, anti-inflammatory agents and anti-bacterial agentswhich are in the main antibiotics. Examples of substances which can beconjugated are given in EP-A No. 2-0114685 and U.S. Pat. No. 4,587,122and include daunomycin, mitomycin, cephalothin, penicillin G andsecretin. Yet further examples of pharmaceutically active substanceswhich may be conjugated with protein fragments to form conjugatesaccording to the present invention are protein growth factors topromote, for example, localised wound repair and antibodies (e.g. of theIgM or IgG class).

The one essential characteristic which is possessed by pharmaceuticallyactive substances suitable for incorporation within conjugates of thepresent invention is their ability to combine with proteins, especiallyadhesive glycoprotein fragments, or non-toxic carrier molecules such asdextran either directly or via a linking group. They may, for example,contain a functional group such as an amino, carboxy or hydroxyl group.One particular active substance of interest for incorporation inconjugates of the present invention is gold, which can be conjugateddirectly with sulphydryl groups present in proteins.

In order to increase the efficiency of delivery of a pharmaceuticallyactive substance to a specified body tissue, a conjugate of the presentinvention may be prepared in which the active substance is loaded on toa non-toxic carrier molecule such as a dextran, preferably a proteinsuch as fibronectin or albumin or a portion thereof, and conjugateddirectly or indirectly with one or more targetting protein fragments.The carrier will generally be covalently conjugated. Clearly, manyadhesive protein fragments, for example from 1 to 20, especially from 1to 5, may be conjugated to a single carrier molecule.

Thus, it will be appreciated that where the chosen carrier isfibronectin or a high molecular weight portion thereof having a range ofdifferent binding specificities, these will be largely negated by theattachment of the active substance and protein fragments and will thusbe prevented from substantially reducing the selectivity of theconjugate. Use of such a carrier is especially preferred for thepreparation of anti-rheumatic conjugates of the present inventionwherein gold or a gold compound is employed together with one or moregelatin-binding fibronectin fragments. Conventional administration of atoxic gold compound for treatment of rheumatoid arthritis has thedisadvantage that undesirable side effects are liable to occur,particularly as a result of accumulation of gold in the liver andkidneys. However, by employing an anti-rheumatic conjugate of thepresent invention wherein gold or a gold compound is bound to both acarrier molecule and at least one targetting fibronectin fragment havinga high degree of selectivity for gelatin binding in the body, a highconcentration of gold can be achieved at arthritic joints with asubstantially reduced risk of liver or kidney damage. Particularlypreferred are conjugates of this type wherein gold per se, derived forexample from aurothiomalic acid or a salt thereof, is directlyconjugated to sulphydryl groups of a carrier protein. In such aconjugate, the targetting protein fragment(s), preferably one or morefibronectin fragments having gelatin-binding affinity, but substantiallylacking fibrin-binding affinity, will be conjugated to the carrier bymeans of a cross-linking agent, e.g. cyanamide. Because of its highcapacity for binding gold via sulphydryl groups, fibronectin or aportion thereof is especially preferred as the carrier molecule for aconjugate of this type. During preparation of such an anti-rheumaticconjugate employing whole fibronectin or a portion thereof having agelatin binding domain, precautionary measures may be taken to protectthe gelatin-binding affinity of the carrier protein of the finalconjugate Thus, direct covalent conjugation of the carrier protein withgold may be carried out in the presence of gelatin, e.g. soluble gelatinor gelatin bound to an agarose-based support such as Sepharose*.Moreover, binding of the targetting protein fragment(s) may be carriedout under mild conditions which do not destroy the gelatin-bindingability of the carrier protein. By taking such protective measures toensure retention of a gelatin binding site in the carrier protein, theselectivity of the final conjugate will not be reduced and indeed it maybe enhanced. ≠* trade mark

The preparation of fragments of an adhesive glycoprotein having enhancedspecificity compared with the parent protein for a single tissue typecan be achieved by adopting the following procedural steps:

(a) Select an adhesive glycoprotein having the desired tissue bindingspecificity;

(b) Fragment the selected protein, preferably by enzymic digestion witha protease, e.g. trypsin, thrombin or cathepsin D; and

(c) Select those fragments which have specific affinity or at least ahigh degree of selectivity compared with the parent protein for the bodytissue involved in the disorder to be treated.

In the case of preparation of a conjugate of the present inventionwherein the pharmaceutically active substance is non-proteinaceous, e.g.gold or a gold compound, it will be understood that the active substancemay be conjugated directly or via a cross-linking reagent to theadhesive protein selected in step (a) prior to proteolysis, theconjugation conditions being chosen so that the adhesive protein retainsbinding affinity for the tissue of interest.

Step (c) is preferably conducted by affinity chromatography. Thus, theprotein fragments resulting from proteolysis in step (b) may besubjected to affinity chromatography on an affinity support havingspecific binding affinity for the tissue binding site of interest or aclosely associated non-tissue binding site. The bound fragments may thenbe eluted and, if necessary, one or more further affinity chromatographysteps subsequently carried out to remove fragments which carry inaddition to the tissue binding site of interest one or more additionaltissue binding sites. The initial affinity chromatography step and anysubsequent affinity chromatography steps may, for example, be carriedout on an affinity support having immobilized thereon appropriate bodytissue or simulated body tissue. For such an affinity support, thetissue or simulated tissue may be conveniently immobilized on anagarose-based support, e.g. Sepharose.

Where the chosen protein for proteolysis has more than one tissuebinding specificity, alternatively in order to obtain fragments havingthe desired enhanced specificity for a particular tissue type, step (c)may begin with subjecting the protein fragments from step (b) to one ormore separation steps in which protein fragments having affinity forbody tissues other than the tissue of interest are selectively removed.Each such separation step may be conveniently performed, for example, byaffinity chromatography on immobilised body tissue (or simulated bodytissue) for which binding affinity is not required in the fragments ofinterest. Finally, the desired fragments may be isolated by affinitychromatography employing a further affinity support with immobilisedbody tissue or simulated body tissue having affinity for the tissuebinding site of interest or by employing an affinity support withspecific binding affinity for a non-tissue binding site closelyassociated with the required tissue binding site.

The size of the selected fragments will depend upon the size of theprotein from which the fragments are generated, the method of proteinfragmentation (which is preferably by proteolytic enzyme digestion), andon the extent and severity of the fragmentation method employed.

One protein from which the fragments can be derived is thenaturally-occurring adhesive glycoprotein fibronectin (m.w. 440,000),which is known to possess a wide range of binding sites, includingbinding sites for gelatin and fibrin, and can be readily digested intofragments by proteolytic enzymes. It is found in plasma and other bodyfluids and is associated with connective tissues, cell surfaces andbasement membranes. Its wide variety of biological functions isattributed to a series of specific binding sites which bind it not onlyto gelatin and fibrin, but also to cell surfaces, glycosaminoglycans andother macromoleculaes. Plasma fibronectin is composed of two verysimilar, but non-identical polypeptide chains which are connected by adisulphide bond at the COOH-terminus and is more susceptible toproteolysis than other basement membrane and plasma proteins. Serineproteases cleave intact fibronectin initially at two preferential sites,releasing a short COOH-terminal fragment containing the interchaindisulphides and an NH₂ -terminal fragment, mw 27,000-30,000. Thiscarries binding sites for fibrin, actin, S. aureus and heparin and across-linking site for plasma transglutaminase (factor XIIIa). Thedigestion proceeds yielding the binding domains shown below:

    ______________________________________                                        Protein(1)(2)(3)(4)(5)(6)(7)                                                  Fragment                                                                       ##STR1##                                                                     ______________________________________                                         Fragment                                                                      1  Binds to fibrin, heparin, actin and S.                                     2  Binds to gelatin, collagens and fibronectin                                4  Binds to cells                                                             5  Binds to heparin                                                           6  Bind to fibrin                                                        

Fibronectin fragments having binding affinity for fibrin in the absenceof gelatin-binding affinity or gelatin-binding affinity in the absenceof fibrin-binding affinity may be conveniently isolated from a mixtureof fibronectin fragments, e.g. a protease digestion mixture of wholefibronectin, by an appropriate two stage affinity chromatographyprocedure employing a fibrin monomer-affinity support, e.g. fibrinmonomer-Sepharose, and a gelatin-affinity support e.g.gelatin-Sepharose. Since the fibrin-binding sites of fibronectin areclosely associated with heparin-binding sites, the fibrinmonomer-affinity support in the above-indicated fragment purificationprocedures may be advantageously substituted by a heparin-affinitysupport, e.g. heparin-Sepharose, which has a higher capacity than fibrinmonomer-sepharose and can be more readily prepared. Indeed,heparin-Sepharose may be obtained from commercial sources, e.g.Pharmacia A.B.

The preferred molecular weight range of fibronectin fragments havingfibrin binding specificity is from 25 to 400 kDa as measured by HPLC(high performance liquid chromatography) or from 25 to 200 kDa asmeasured by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gelelectrophoresis), whereas the molecular weight range of fibronectinfragments which have gelatin binding specificity is preferably in therange 40 to 500 kDa as measured by HPLC or 40 to 200 kDa as measured bySDS-PAGE, the smaller fragments in these ranges having increasedspecificity. The higher molecular weights recorded when using HPLC areprobably due to association of fragments before or during measurementwhich give rise to an increase in apparent molecular weights.

Thus, according to a further aspect of the present invention, we providea method of preparing a pharmaceutically active conjugate of the presentinvention which comprises conjugating a pharmaceutically activesubstance directly or indirectly to at least one fragment of an adhesiveglycoprotein having improved binding specificity for a body tissueinvolved in the disorder to be treated compared with the whole adhesiveprotein or, where the pharmaceutically active substance is not aprotein, conjugating the pharmaceutically active substance to the wholeadhesive glycoprotein or a portion thereof, optionally with protectionof binding sites specific to the said body tissue, followed byproteolysis to produce protein fragments carrying conjugatedpharmaceutically active substance and selection of at least one of saidfragments having improved binding specificity compared with the saidprotein or portion thereof.

Thus, for example, an anti-rheumatic conjugate according to the presentinvention may be prepared by directly conjugating gold to sulphydrylgroups of an adhesive protein possessing gelatin-binding affinity, e.g.whole fibronectin, followed by protease digestion and selection of aconjugate with improved selectivity over the parent conjugate forgelatin-binding in the body. Where fibronectin or a gelatin-bindingportion thereof is chosen as the starting adhesive protein, goldconjugation will be carried out in the presence of gelatin, e.g. solublegelatin or gelatin-bound to an agarose-based support, so as to protectthe gelatin-binding site of the adhesive protein.

In the case of a process according to the present invention where one ormore fragments of an adhesive protein having improved bindingspecificity compared with the parent protein for the tissue of interestare employed for conjugation to a pharmaceutically active substance, thepharmaceutically active substance may be bound, preferably covalently,to a carrier prior to conjugation directly or indirectly with thetargetting protein fragment(s) or such carrier binding may be carriedout subsequent to protein fragment binding, in which case conditionswill be chosen so that the required tissue binding capability of theprotein fragment(s) is retained. Where a conjugate of the presentinvention comprising as the pharmaceutically active substance anon-proteinaceous species is generated by protease digestion of a largeradhesive protein-containing conjugate, it may also be feasible tosubsequently bind the selected conjugate to a carrier withoutsubstantially reducing the desired tissue binding specificity.

The activity of conjugates of the present invention increases withincreasing loading of active substance bound to the protein fragment(s)and so the active substance is preferably present in molar excess withinthe conjugate. However, too high a loading of active substance will tendto mask the required tissue binding site on the tissue-binding substrateand may give rise to poor conjugation efficiency. For this reason, aconjugate of the present invention preferably contains from 1 to 100,more preferably from 1 to 50, moles of active substance (usually asingle compound or element) per mole of protein fragment. When theactive substance consists of a protein or enzyme or other high molecularweight substance, the molar ratio of protein fragment to activesubstance in the conjugate is preferably from 1:1 to 1:10. On the otherhand, active substances of low molecular weight, for example gold atoms,are preferably present at higher loading ratios, for example 1:10 to1:50.

In the preparation of a conjugate according to the present invention,other than a gold-protein fragment anti-rheumatic conjugate ashereinbefore described, the protein fragment or fragments are mostpreferably bound with the pharmaceutically active substance using aknown protein cross-linking agent such as a carbodiimide, a dialdehydoderivative of a dicarboxyliic acid, a diisocyanate, or an oxidiseddextran having an aldohexopyranose ring-cleaved structure.

Suitable carbodiimides include any of those disclosed in U.S. Pat. No4,046,871 and may be selected from any of the following:

1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride,

1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimidemetho-p-toluene-sulphonate,

1-cyclohexyl-3-(4-diethylaminocyclohexyl) carbodiimide metho-ptoluene-sulphonate,

1-cyclohexyl-3-(β-diethylamino-ethyl)-carbodiimide,

1-ethyl-3-(2-morpholino-ethyl)-carbodiimide hydrochloride,

1-ethyl-3-(2-morpholino-ethyl)-carbodiimide sulfate and cyanamide.

Where the cross-linking agent employed consists of a dialdehydoderivative of a dicarboxylic acid, the acid is preferably acarboxy-terminated C₂ -C₅ straight chain alkane and is most preferablyglutaric acid, the dialdehydo derivative of which is glutaraldehyde. Asuitable method of conjugation employing glutaraldehyde is given by J.W. Payne in Biochem J (1973) 135, 867-873.

An example of a suitable diisocyanate is hexamethylene diisocyanate.

Suitable oxidised dextrans and methods of conjugation employing the sameare disclosed in U.S. Pat. No. 4,587,122.

Covalent coupling of a pharmaceutically active substance with one ormore protein fragments for preparation of a conjugate of the presentinvention may, for example, be carried out by (a) mixing and reactingsimultaneously the protein fragment(s), cross-linking agent andpharmaceutically active substance, (b) reacting the cross-linking agentwith the pharmaceutically-active substance, and then reacting theproduct with the protein fragment(s), or (c) reacting the cross-linkingagent with the protein fragment(s), and then reacting the product withthe pharmaceutically active substance.

Covalent coupling of a pharmaceutically active substance with one ormore protein fragments to form a conjugate of the present invention willgenerally be conducted in an aqueous solution of pH 5 to 9, preferably 6to 8, and preferably in a buffer solution. Preferred reactiontemperatures are 10° C. to 30° C., particularly room temperature. Thereaction time is preferably from 0.5 to 15 hours, more preferably from0.5 to 5 hours. The preferred molar ratio of pharmaceutically activesubstance to protein fragment in the reaction mixture is preferably from1:1 to 100:1, with, if appropriate, sufficient or excess cross-linkingagent being present to effect conjugation of the two.

Active substance-protein fragment conjugates produced by the methodsdescribed above are preferably separated from the reaction mixture inwhich they are produced by affinity chromatography on immobilised bodytissue or simulated body tissue for which the protein fragments haveaffinity.

Subsequent covalent coupling of an active substance protein fragmentconjugate to a high molecular weight carrier, e.g. fibronectin, albuminor a high molecular weight portion thereof, may also, for example, becarried out by employing a cross-linking reagent under mild conditionsas specified above. Preferably, however, when it is desired to prepare aconjugate according to the present invention with a carrier, the carrierwill be covalently coupled to the pharmaceutically active substance,either directly or via a coupling reagent, prior to protein fragmentbinding. Thus, for preparation of a preferred anti-rheumatic conjugateof the present invention wherein gold is conjugated to a carrierprotein, an appropriate gold compound, e.g. aurothiomalic acid or a saltthereof such as sodium aurothiomalate, will preferably initially bereacted with the sulphydryl groups of the carrier so as to directlycouple gold via these groups. As hereinbefore indicated, where theprotein chosen for this step is fibronectin or a gelatin binding portionthereof, the carrier conjugation may be carried out in the presence ofgelatin, e.g. soluble gelatin or gelatin bound to an agarose basedsupport, e.g. Sepharose, in which case the carrier protein will retain agelatin-binding site. Alternatively, fibronectin may, for example,conveniently be directly coupled with gold when conjugated itself to anagarose-based support such as Sepharose or when adsorbed on tofibronectin thus bound. For the preparation of a carrier containing,anti-rheumatic conjugate of the present invention, gold may alsoconveniently be directly conjugated to albumin via sulphydryl groups ofthe protein by, for example, reacting an appropriate gold compound suchas sodium aurothiomalate with albumin bound to an agarose-based support,e.g. an albumin-Sepharose column. If in the preparation of ananti-rheumatic conjugate of the present invention gold is initiallyconjugated to a protein which is itself covalently conjugated on asupport, e.g. Sepharose, it will be understood that the requiredgold-carrier protein conjugate may be released by appropriate proteasedigestion, i.e. the carrier protein of the final conjugate will bederived from a larger pre-carrier protein.

According to a third aspect of the present invention, there is provideda pharmaceutical composition which comprises a pharmaceutically activeconjugate according to the first aspect dissolved or dispersed in apharmaceutically acceptable diluent or carrier, for example salinesolution. Administration of the composition may be by intravenousinjection or by oral ingestion of the composition in the form of atablet or ingestible .liquid. A typical dose of aqueous composition maycontain 10-300 mg of conjugate in 0.05-10 ml of composition.

According to a further aspect of the present invention, we provide aconjugate according to the present invention for use in therapeutictreatment of a human or non-human animal, e.g. conjugates of the presentinvention wherein a plasminogen activator is bound to a fibronectinfragment having predominantly fibrin targetting capability for use intreatment of a thrombotic condition or use of a conjugate according tothe present invention wherein an anti-rheumatic substance, e.g. gold, isconjugated to a fibronectin fragment having predominantly gelatintargetting capability for use in the treatment of rheumatoid arthritis.

We also provide as a still further aspect of the present invention, useof a conjugate according to the present invention for the preparation ofa composition for use in the treatment of a disorder involving aspecified body tissue for which said conjugate has predominanttargetting capability.

As yet another aspect of the present invention, we additionally providea method of delivery of a pharmaceutically active substance to a tissueinvolved in a disorder of the body wherein said pharmaceutically activesubstance is administered in the form of a conjugate according to thepresent invention having predominant targetting capability for saidtissue.

The following non-limiting examples are intended to illustrate thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the correlation between the amount of conjugate added tothe immobilized physiological fibrin monomer (PFM) and the amount ofplasminogen activator (PA) subsequently bound to it.

FIG. 2 shows the relationship for conjugates prepared by the proceduresE and G-I between the amount of PA (in units) per mg fibronectinfragment used in the cross-linking reaction mixture that produced theconjugates and the amount of fibrin-binding PA which was produced in theconjugates.

FIG. 3 shows the time course of ¹²⁵ I radioactivity released from fibrinclot discs in response to urokinase activity.

EXAMPLE 1 PREPARATION OF ANTI-THROMBOTIC CONJUGATES A. Materials Al.Preparation of Physiological Fibrin Monomer (PFM)

The PFM used in the following examples consisted of fibrinogen which waspurified by precipitation from blood plasma cryoprecipitate supplied bythe Plasma Fractionation Laboratory, Churchill Hospital, Oxford, England(GB). This PFM contained 68 wt.% fibrinogen, 10% fibronectin (a naturalcontaminant) with traces of factor VIII:RAg and factor XIIIa. Sincenormal fibrin blood clots invariably incorporate fibronectin, this PFMwas subsequently used as a simulated fibrin blood clot.

A2. Preparation of Fibronectin-Free Fibrin Monomer (FFFM)

FFFM was prepared by removing fibronectin from the fibrinogen within PFMby subjecting the fibrinogen to gelatin-sepharose affinitychromatography. The preparation of columns used in this chromatographicprocedure is described below.

B. General Procedures B1. Preparation and Use of Gelatin-SepharoseAffinity Chromatography Columns

Gelatin-Sepharose columns were prepared following the coupling procedureoutlined in the manufacturer's (Pharmacia, Uppsala, Sweden) recommendedprocedure for adsorbing materials on to CNBr-activated Sepharose 4B. Acoupling ratio of 15 mg gelatin per g moist weight Sepharose gel wasused to prepare the columns.

The running conditions for the prepared columns were adapted from Vuentoand Vaheri, J. Biochem (1979) 183, p.331 with the following changes:-therunning buffer consisted of a 10 mM phosphate, 10 mM citrate, 150 mMNaCl solution pH 7.5 and elution was achieved with 1 M arginine inphosphate buffered saline. The columns were used at 4° C.

B2. Preparation and Use of Fibrin Monomer-Sepharose AffinityChromatography Columns

Fibrin monomer columns were prepared following the method of Heene andMatthias, Throm. Res. (1973) 2, p.137. Two types of column wereprepared, in which the monomer used was either Physiological FibrinMonomer (PFM) or Fibronectin Free Fibrin Monomer (FFFM). PFM--Sepharoseand FFFM--Sepharose columns were prepared by coupling either PFM or FFFMto CNBr-activated Sepharose 4B by the manufacturer's (Pharmacia)recommended procedure. The running conditions for both types of columnwere the same as those used in General Procedure B1 described above.

B3. Preparation and use of Heparin-Sepharose Affinity ChromatographyColumns

Heparin-Sepharose columns were prepared following the procedurerecommended by the manufacturer for coupling materials on toCNBr-activated Sepharose 4B. Such columns are also available`ready-coupled` from the same manufacturer (Pharmacia, Uppsala, Sweden).Running conditions were as described above for gelatin-Sepharose, exceptthat the running buffer consisted of a 10 mM phosphate, 20 mM NaCl, 0.5mM EDTA solution, pH 7.5 and elution was achieved using a 10 mMphosphate, 0.5 M NaCl, 0.5 mM EDTA solution, pH 7.5. Columns were usedat room temperature.

B4. Separation of Protein Fragments on Sephacryl*S200

Further resolution of protein fragments selected by affinitychromatography was carried out by gel permeation chromatography usingSephacryl S200. The running buffer was 10 mM phosphate, 150 mM NaCl, pH7.5. Approximately 10 mg were applied to a 320 ml bed volume column(2.2×84 cm) at a flow rate of 12 ml/hr. ≠* trade mark

B5. Assay for Plasminogen Activators

The concentrations of plasminogen activator in solutions were determinedusing the Kabi Diagnostica "Initial Rate of Reaction" method for theDetermination of Plasminogen in Plasma, using S-2251 chromogenicsubstrate (Kabi Vitrium, Sweden). The protocol for this method issummarised below:

(1) Dilute human blood plasma with assay buffer solution consisting of50 mM Tris-HCl, pH 7.4 containing 12 mM NaCl in the volumetric ratio ofplasma to buffer solution of 1:20 and add 200 microliters of the dilutedplasma to a reaction tube.

(2) Incubate the tube at 37° C. for 2 to 6 minutes.

(3) Add either a known number of units (e.g. 10 or 25 units) ofplasminogen activator or a test conjugate containing plasminogenactivator, made up to 100 microliters with assay buffer solution, to thetube.

(4) Incubate the tube at 37° C. for 10 minutes.

(5) Add 700 microliters of substrate solution consisting of S-2251diluted to 0.86 mM working solution with assay buffer.

(6) Mix, transfer the contents of the tube to a micro-cuvette andmeasure the change with time of the absorbance (A) of the mixture at 37°C. to 405 nm wavelength light (ΔA₄₀₅).

(7) Calculate ΔA₄₀₅ per minute and plot this result against the units ofplasminogen activator per ml present in the buffer solution.

An alternative plasminogen activator assay was used to test for suchactivity on PFM - Sepharose. An end-point assay had to be used becauseSepharose could not be tested by the "initial rate of reaction" assaydescribed above. The assay protocol is summarised below:

(1) Partly dry the test PFM - Sepharose by suction filtration on asinter funnel.

(2) Weigh 100mg of test PFM - Sepharose (moist weight) into a reactiontube.

(3) Add 200 microliters of diluted human blood plasma.

(4) Mix

(5) Incubate the tube for 10 minutes at 37° C.

(6) Add 700 microliters of S-2251 chromogenic substrate (Kabi) solutionat 37° C., mix and incubate for exactly 180 seconds.

(7) Add 100 microliters of 50% acetic acid and mix immediately to stopthe reaction.

(8) Measure the absorbance of the resulting solution to 405 nm within 4hours and relate the result to a standard curve for known concentrationsof plasminogen activator against absorbance.

C. Generation Of Protein Fragments By Digestion Of An Adhesive ProteinWith Proteolytic Enzymes

Fibronectin was chosen as the starting adhesive protein. Prior todigestion, the fibronectin was checked for purity by size exclusion HighPerformance Liquid Chromatography (HPLC) using an Ultropac TSK G 4000 SWcolumn, fractionation range from 1000kDa down to 5kDa. This wasnecessary in order to standardise the fibronectin fragments generated byproteolytic enzyme digestion, since the presence of fibronectinaggregates or fragments in the fibronectin prior to digestion could haveaffected the subsequent digestion process.

C1. Digestion of fibronectin with trypsin

10 mg of fibronectin in 10 ml of PBS (PBS =phosphate buffered saline pH7.5 containing 10 mM sodium phosphate and 0.15 M NaCl) was digested with0.05 mg of trypsin for 2 hours at 37° C. Trypsin is an endopeptidasecapable of specifically cleaving peptide bonds adjacent to an arginineor lysine residue. Samples of the reaction mixture were removed at thetimes stated in Table 1 below. The reaction was stopped after 2 hrs bythe addition of 0.1 mg of soya bean inhibitor.

The molecular weights of the fibronectin fragments produced by digestionwere determined by gel permeation high performance liquid chromatography(HPLC) analysis using a TSK G 3000 SW column. The running buffer was a0.1 M phosphate solution pH 6.8 containing 0.05% sodium azide, flow rate0.4 ml/min, with a 70 minute elution time. The absorbance of the eluatewas monitored at 280 nm for the presence of fibronectin fragments.

                  TABLE 1                                                         ______________________________________                                        Digestion of Fibronectin with Trypsin                                         Incubation time                                                               (minutes)  Protein Fragment Molecular Weight (kDa)                            ______________________________________                                        0          439     --      --    --    --   --                                10         --      358     --    140   56   42                                30         --      358     181   140   56   42                                60         --      358     181   140   56   42                                120        --      --      181   140   56   42                                ______________________________________                                    

Extensive digestion of fibronectin by trypsin (that is, for more than 10minutes) dramatically reduced its gelatin binding activity, as measuredby a competitive, enzyme-linked assay (Doran et al, Vox Sang (1983) 45,243-251). Consequently, the principal of using the minimum effectivedigestion time of 10 minutes was adopted.

C2. Digestion of fibronectin with thrombin

The procedure of C1 was repeated, except that the fibronectin wasdigested with 250 international units (iu) of thrombin instead oftrypsin. Thrombin is a serine protease responsible for the finalconversion of fibrinogen to fibrin during blood clot formation. Thedigestion was stopped after 120 minutes by the addition of benzamidineto a final concentration of 8mM in the reaction mixture. Samples of thereaction mixture were removed at the times stated in Table 2 below andwere analysed using the same procedure as outlined in C1.

                  TABLE 2                                                         ______________________________________                                        Digestion of Fibronectin with Thrombin                                        Incubation time                                                               (minutes)  Protein Fragment Molecular Weight (kDa)                            ______________________________________                                        0          439     --      --    --    --   --                                10         468     218     83    --    --   --                                30         462     218     83    --    --   --                                60         468     --      83    50    27   23                                120        470     218     110   45    28   --                                ______________________________________                                    

D. Selection and Purification of Protein Fragments

Products of fibronectin digestion with trypsin or thrombin comprising amixture of fragments with either or both gelatin-binding affinity andfibrin-binding affinity or neither of these two binding specificitieswere used as the starting material to select out fibronectin fragmentshaving fibrin-binding affinity in the absence of gelatin-bindingaffinity or vice versa using a dual column affinity chromatographyprocedure.

D1. Use of thrombin generated fibronectin fragments

The method of C2 was repeated on a larger scale with the quantities ofreagents and solution volume increased proportionally. The product,consisting of a solution of fibronectin fragments obtained byfibronectin digestion for 120 minutes at 37° C. with thrombin, wasdivided into several aliquots containing known amounts of fibronectinfragments. These aliquots were applied directly to a 15 ml column ofgelatin-Sepharose (see the general procedure B1 described above) inorder to remove those protein fragments with an affinity for gelatinpossessing both gelatin and fibrin-binding sites or a gelatin bindingsite in the absence of a fibrin-binding site. The unbound fractions ofthese aliquots, which contained protein fragments with an affinity forfibrin in the absence of gelatin-binding affinity and protein fragmentshaving neither fibrin nor gelatin-binding affinity, were then applied toa 15 ml column of FFFM-Sepharose (see the general procedure B2 describedabove). The eluates from the FFFM-Sepharose column contained purifiedprotein fragments with binding affinity for fibrin but no bindingaffinity for gelatin. The results of this two-stageaffinity-chromatography procedure are given in Table 3 below in terms ofproduct yields.

                                      TABLE 3                                     __________________________________________________________________________    2-stage affinity chromatography of fibrin-binding protein fragments           Sample                                                                             Gelatin-Sepharose  FFFM-Sepharose                                        Applied                                                                            Affinity Chromatography                                                                    % Protein                                                                           Affinity Chromatography                                                                    % Protein                                (mg) mg bound                                                                            mg unbound                                                                           Recovery                                                                            mg bound                                                                            mg unbound                                                                           Recovery                                 __________________________________________________________________________    33.0 2.84  22.94  78    1.19  3.79   11.5                                     15.0 6.4   6.5    86    1.72  2.44   68.4                                     7.2  2.64  2.56   72    0.74  1.40   81.0                                     __________________________________________________________________________

The recoveries of protein fragments at each stage given in Table 3 abovewere based on the absorbance of fractions at 280 nm using the extinctioncoefficient for fibronectin ##EQU1##

The eluates from the FFFM-Sepharose and gelatin-sepharose columns wereanalysed by HPLC to determine the molecular weights of both gelatinbinding and fibrin binding fragments. The sizes of fragments eluted fromthe gelatin column were found to be 435 and 296 kDa. The sizes of thefragments eluted from the FFFM column were found to be 382, 110 and 45kDa.

It will be understood that the above 2-stage affinity chromatographyprocedure can be reversed (FFFM-adsorption followed by gelatinadsorption) to afford eluates from the second adsorption stage thatcontain purified protein fragments with binding affinity for gelatin inthe absence of fibrin binding affinity.

D2. Use of Trypsin generated fibronectin fragments

(a) The procedure of D1 was repeated using fibronectin digested for 10minutes with trypsin in a larger scale version of the proceduredescribed in C1 above. The eluates from the affinity columns wereanalysed by HPLC. This revealed that protein fragments were eluted fromthe gelatin column of sizes 358, 140, 119 and 69 kDa and proteinfragments of 56 kDa were eluted from the FFFM column.

(b) The close association between the fibrin binding sites and heparinbinding sites in fibronectin was exploited by using heparin-Sepharoseinstead of FFFM-Sepharose in an alternative dual column affinitychromatography procedure to isolate fibronectin fragments havingfibrin-binding affinity in the absence of gelatin-binding affinity.

A solution of tryptic fragments of fibronectin was prepared as in D2(a)above and the solution divided into several aliquots containing knownamounts of fibronectin fragments. Aliquots were applied to a 15 mlcolumn of heparin-Sepharose by the general procedure previouslydescribed in B3 above. Bound fractions which contained protein fragmentswith an affinity for heparin and fibrin were then applied to a 30 mlcolumn of gelatin-Sepharose by the procedure described in B1 Unboundfractions contained purified protein fragments with an affinity forheparin/fibrin. Those binding to gelatin-Sepharose, which were thosewith both gelatin and heparin/fibrin binding activity, were discarded.Results of this two stage affinity chromatography procedure are given inTable 4 below.

The eluates from the adsorbent columns were analysed by HPLC. Thisrevealed that the sizes of the protein fragments from the two-stagepurification procedure were 360, 181, 140 (minor), 56 and 42 kDa.

While all of these fragments retained heparin/fibrin binding activityand did not bind to gelatin, the different fragment sizes generated bythis method could be further resolved by separation on Sephacryl S200 asdescribed in B4 above. The proportions of the fragments in the heparinbinding, non-gelatin binding fraction are given in Table 5.

The proportion of protein fragments at each stage which werefibrin-binding was determined by applying aliquots to a PFM-Sepharosecolumn by the procedure of B2 above.

                                      TABLE 4                                     __________________________________________________________________________    Sample                                                                             Heparin-Sepharose  Gelatin-Sepharose                                     Applied                                                                            affinity chromatography                                                                    % Protein                                                                           Affinity Chromatography                                                                    % Protein                                (mg) mg bound                                                                            mg unbound                                                                           Recovery                                                                            mg bound                                                                            mg unbound                                                                           Recovery                                 __________________________________________________________________________    23.7 11.2  13.7   105.0 1.8   9.1    97.3                                     6.0  3.22  2.72   99.0  0.32  2.85   98.4                                     __________________________________________________________________________

                  TABLE 5                                                         ______________________________________                                                       Recovered           % Fibrin-                                                 Protein   % Starting                                                                              binding                                    Purification Stage                                                                           (mg)      material  activity                                   ______________________________________                                        Fibronectin trypsin digest                                                                   23.7      100            23                                    Heparin-Sepharose                                                                            11.12     45.6                                                 (bound fractions)                                                             Gelatin-Sepharose                                                                            9.10      37.3           63                                    (unbound fraction)                                                            Sephacryl S200                                                                Fractions:                                                                    360K           2.61      11.0           23                                    181K           1.49      6.3            39                                    (140K - minor peak)                                                                          N.D.      N.D.                                                  56K            1.75      7.4                                                                                         68                                    42K            1.19      5.0                                                  ______________________________________                                         N.D. -- Not determined                                                   

It will be appreciated that if the two types of affinity chromatographycolumn are used in opposite order, i.e. the bound fraction of agelatin-Sepharose column is applied to a heparin-Sepharose column, thiswill result in isolation of fibronectin fragments having gelatin-bindingaffinity in the absence of fibrin and heparin binding affinity.

E. Conjugation of selected fibronectin fragments having fibrin-bindingaffinity to streptokinase

An aqueous solution of a commercial streptokinase preparation (tradename "Kabikinase" supplied by Kabi Vitrum AB Haematology, Stockholm,Sweden) containing by weight 4% streptokinase, 50% albumin, 44% sodiumsalts and 2% H₂ O was passed through a column of Cibacron Blue-SepharoseCL6B adsorption resin at ambient temperatures in order to adsorb thealbumin, thereby separating it from the streptokinase. The unboundfractions were assayed for plasminogen activator activity.

2 mls of an aqueous solution of the unbound fractions, containing about600 units of albumin-depleted streptokinase per ml, were prepared by theprocedure described above. An equal volume of an aqueous solution offibrin-binding fibronectin fragments prepared in accordance with themethod of D1 (0.12 mg per ml) was added to the streptokinase solution.The pH of the mixture was adjusted to 7.0 by the addition of a smallamount of 0.1 N hydrochloric acid. To the resulting solution was added,with stirring and at ambient temperature, a 0.588 M1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride solutionuntil the content of the carbodiimide hydrochloride in the resultingsolution reached 0.5% (w/v). Stirring was continued for 2 hours toproduce a conjugated streptokinase-protein fragment product in thereaction mixture. The desired product was recovered from the mixture byadsorption on to FFFM-Sepharose followed by elution with 0.2M NaClsolution. The concentration of the conjugate in the resulting eluatewas, where necessary, increased by pressure ultra-filtration.

The activity of the streptokinase within the conjugate at variousconjugate concentrations in solution was measured by the proceduredescribed in B5 above against equivalent concentrations in solution ofunconjugated streptokinase. The results of these measurements are givenin Table 6 below.

                  TABLE 6                                                         ______________________________________                                        SK            Activity (Δ A.sub.405 min.sup.-1)                         concentration          SK-protein                                             (units/ml)    SK alone fragment conjugate                                     ______________________________________                                        10            0.019    0.010                                                  20            0.036    0.014                                                  30            0.053    0.021                                                  ______________________________________                                    

These results show that streptokinase activity in the conjugate variedfrom 38%-52% of that present in its unconjugated form.

F. Conjugation of selected fibronectin fragments having fibrin-bindingaffinity to urokinase

2240 units of urokinase (UK) were dissolved in 2 ml of pH 7.3 buffersolution (20mM Tris HCl, 0.15 M NaCl) containing 0.12 mg/ml offibrin-binding fibronectin fragments prepared in accordance with themethod of D1. The total content of protein fragments within the solutionwas equivalent to 3.27×10⁻⁶ M. To the resulting solution was added 80microliters of 5% (w/v) glutaraldehyde solution in water (finalconcentration 0.2%). The resulting solution was mixed on a vortex mixerwhile the glutaraldehyde was added. Mixing was continued for 1 minuteand the solution was then incubated at room temperature (20° C.) for 24hours. The desired product was recovered using FFFM-Sepharose asdescribed in procedure E above. The concentration of the conjugate insolution was adjusted as necessary either by pressure ultrafiltrationconcentration or by dilution with buffer solution.

The activity of the urokinase within the conjugate at various conjugateconcentrations in solution was measured by the procedure described in B5above against equivalent concentrations in solution of unconjugatedurokinase. The results of these measurements are given in Table 7.

                  TABLE 7                                                         ______________________________________                                        UK           Activity (Δ A.sub.405 min.sup.-1)                          concentration          UK-protein                                             (units/ml)   UK alone  fragment conjugate                                     ______________________________________                                        10           0.020     0.006                                                  20           0.0475    0.010                                                  30           0.0575    0.013                                                  ______________________________________                                    

The results given in Table 7 above show that the plasminogen activity ofthe urokinase within the conjugate is 21%-30% of its activity when inits original, unconjugated form.

G. Alternative method for conjugation of selected fibronectin fragmentshaving fibrin-binding affinity to urokinase

2240 units of UK were dissolved in 2 ml of pH 7.3 buffer solution (20mMTris-HCl, 0.15M NaCl) containing 0.12 mg/ml (3.27×10⁻⁶ M) offibrin-binding fibronectin fragments prepared as in D1 above. The pH ofthe solution was adjusted to 5 by the addition of small amounts of 0.1 Nhydrochloric acid. To the resulting solution was added, with stirringand at ambient temperature, a 0.588 M1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride solutionuntil the content of the carbodiimide hydrochloride in the resultingsolution reached 1.0% (w/v). Stirring was continued for 1 hour toproduce a conjugated urokinase-fibronectin fragment product in thereaction mixture. A mild form of carbodiimide cross-linking was alsosuitable using 0.5% (w/v) carbodiimide for 2 hours at pH 7.0 (i.e. noHCl added). The desired conjugated product was recovered from themixture by adsorption on to FFFM-Sepharose followed by elution with 0.2M NaCl solution. The concentration of the conjugate in the resultingeluate was, where necessary, increased by pressure ultrafiltration.

The activity of urokinase within the conjugate at various conjugateconcentrations in solution was measured as in the procedure F above. Theresults of these measurements are given in Table 8 below.

                  TABLE 8                                                         ______________________________________                                        UK           Activity (Δ A.sub.405 min.sup.-1)                          concentration          UK-protein                                             (units/ml)   UK alone  fragment conjugate                                     ______________________________________                                        10           0.0225    0.015                                                  20           0.050     0.033                                                  30           0.060     0.042                                                  ______________________________________                                    

It may be seen from Table 8 above that the plasminogen activatoractivity of the UK within the conjugate varies from 64%-67% of itsactivity when in its original, unconjugated form.

H. Conjugation of selected fibronectin fragments having fibrin-bindingaffinity to urokinase

The method of G above was repeated except that the concentration offibrin-binding fibronectin fragments in the Tris-HCl-saline solution wasreduced to 0.06 mg/ml.

I. Conjugation of selected fibronectin fragments having fibrin-bindingaffinity to urokinase

The method of G above was repeated except that the concentration offibrin-binding fibronectin fragments in the Tris-HCl-saline solution wasreduced to 0.03 mg/ml.

J. Binding plasminogen activator-protein fragment conjugates tosimulated blood clot tissue (fibrin Monomer)

In order to test the efficiency of the conjugates prepared by theprocedures described in E and G-I above, solutions of each conjugatewere contacted with immobilised simulated blood clot tissue and theamount of plasminogen activator adsorbed by the tissue was measured. Itis known that there is a direct correlation within the body between theuptake of plasminogen activator by fibrin clots and their rate ofsubsequent fibrinolysis and so this test provides a useful indication ofthe pharmacological value of these conjugates.

The simulated tissue selected was physiological fibrin monomer (PFM)preparation which is known to include many of the components of a normalblood clot. Crude fibrin monomer (Kabi Diagnostica), herein referred toas PFM, was immobilised on to Sepharose 4B beads (Pharmacia, Sweden) bythe procedure described in B2 above. Plasminogen activator-fibronectinfragment conjugates were made up into separate solutions of knownconcentration containing from 16 to 160 units/ml of plasminogenactivator activity. These solutions were then roller mixed for 2 hoursat 4° C. with 100 mg (moist weight) quantities of the immobilised PFMdescribed above. The immobilised PFM was then washed thoroughly with 50mM TrisHCl solution, pH 7.6 to remove any unbound activity. The quantityof bound conjugate was then determined by direct assay of thePFM-Sepharose using the end point assay described in B5 above andaliquots of the affinity adsorbent in suspension. The activity of theplasminogen activator in both the starting material (plasminogenactivator-fibronectin fragment conjugate) and in the washedPFM-Sepharose after binding was determined (in terms of units ofplasminogen activator (PA) per ml in solution) as a measure offibrin-binding fibrinolytic agent in the test conjugates. The results ofthese determinations are given in Table 9 below.

                                      TABLE 9                                     __________________________________________________________________________    conjugate                                                                           Units/ml of PA                                                          preparation                                                                         within conjugate added                                                                    Units/ml of PA                                                                          % of PA activity                                                                        Units of PA reacted per                 procedure                                                                           to immobilised PFM                                                                        bound to Sepharose                                                                      bound to Sepharose                                                                      mg of protein fragment                  __________________________________________________________________________    E     16          0.42      2.6       133                                           32          3.2       10.0      267                                           80          23.0      28.0      667                                           160         41.6      26.0      1333                                    G     25          6.0       23.6      208                                           50          10.4      20.7      417                                           100         15.3      15.3      833                                           150         17.8      11.9      1250                                    H     50          18.13     36.3      833                                           100         32.0      32.0      1667                                          150         31.6      21.1      2500                                    I     50          25.6      51.2      1667                                          100         34.2      34.2      3111                                          150         42.9      28.6      5000                                    __________________________________________________________________________

The results of Table 9 are illustrated diagrammatically in FIGS. 1 and2. FIG. 1 is a graph showing the correlation between the amount ofconjugate added to the immobilised PFM and the amount of PA subsequentlybound to it. FIG. 2 illustrates the relationship for conjugates preparedby the procedures of E and G-I above between the amount of plasminogenactivator (in units) per mg fibronectin fragment used in thecross-linking reaction mixture that produced the conjugates and theamount of fibrin-binding plasminogen activator which was produced in theconjugates. Using urokinase there is a two-phase slope to the increasein conjugate PA content as more plasminogen activator was employedwithin the cross-linking reaction mixture. The slope with streptokinaseis much steeper indicating more effective binding.

K. Binding of Plasminogen activator-fibronectin fragment conjugates toSepharose 4B

In order to establish that the plasminogen activator-fibronectinfragment conjugates were binding selectively to the fibrin monomerrather than to its support matrix, the product of method G above wasapplied to unsubstituted Sepharose 4B gel using the binding procedureoutlined in J. The resultant fractions were assayed for plasminogenactivator activity. The only activity to be found was in the unboundfractions. Recovery was 100%. Since the conjugate of method G hadnegligible affinity for unsubstituted Sepharose, it was concluded that afibrin-substituted Sepharose behaved as a simulated fibrin clot and theconjugate bound entirely to the protein portion of the simulated clot.

L. Binding of unconjugated plasminogen activator to simulated blood clottissue

2016 units of urokinase were made up into 2 ml of pH 7.3 buffer solution(20 mM Tris HCl containing 0.15 M NaCl) and applied to PFM-Sepharose 4B.The mixture was held at 4° C. for 2 hours. It was found that only 5% ofthe original UK activity present in solution became bound to thesimulated clot, indicating that basal binding of unmodified urokinase tothe simulated blood clot tissue was minimal.

M. Dissolution of fibrin-based clot tissue in response to plasminogenactivators and plasminogen activator-fibronectin fragment conjugates

The pharmacological efficacy of the plasminogen activator-fibronectinfragment conjugate product of method G was further tested by examiningthe lysis of fibrin-based clots in response to the selective binding ofthe conjugate. The influence of targetted plasminogen activatorconjugates was assessed by measurement of ¹²⁵ I-FDPs released from ¹²⁵I-labelled clots into the clot bathing medium.

Fibrinogen was iodinated by the chloramine T method (Green et al,Biochem. J., (1963), 89, p.114) and activated using 100 U thrombin pergram fibrinogen. PBS - washed clot tissue was blotted into the form of aprotein sheet from which small discs (10-20 mg) could be readilyexcised. ¹²⁵ I-Fibrin clot discs (20 mg) were incubated for 2hrs at roomtemperature in 0.25 ml, Tris buffer solution, pH 7.4 (50 mM Tris, 110 mMNaCl) containing either 800 U/ml urokinase or 800 U/mlurokinase-fibronectin fragment conjugate.

The clot discs were washed twice with 2 ml of Tris buffer solution, pH7.4 to remove non-specifically bound urokinase. Control clot discs wereincubated in Tris buffer solution, pH 7.4, without urokinase orconjugate and washed in parallel with test samples.

Clot discs were transferred to a chamber (10 ml vol.) through which theclot bathing medium (25 ml) was circulated from a reservoir at a flowrate of 2 ml/min. The bathing medium consisted of Tris buffer solution,pH 7.4, containing 0.03mg/ml plasminogen and 1 mg/ml human albumin.

Samples (0.5 ml) of the bathing medium were taken at intervals over a 24hr period and solubilised radioactivity determined. The results of thesemeasurements are represented diagrammatically in FIG. 3, which is agraph showing the time course of ¹²⁵ I radioactivity released fromfibrin clot

of discs in response to urokinase activity. Dissolution of the clotsincubated with conjugate was up to 3.3 times faster (for example at 18hrs: control =nil activator-stimulated lysis) than that with theequivalent activity of plasminogen activator alone. This indicated thatspecific and preferential binding of conjugate can enhance theactivation of plasminogen in the vicinity of a clot and hencefibrinolytic breakdown of the clot.

EXAMPLE 2 PREPARATION OF ANTI-RHEUMATIC CONJUGATES A. Direct CovalentCoupling of Gold to Fibronectin or Albumin A1. Direct covalent couplingof gold to fibronectin fibronectin-Sepharose or albumin-Sepharose

1 ml of fibronectin-Sepharose slurry [1-2 parts coupling buffer (50 mMTris-HCl, pH 7.5, 1 M urea) plus 1 part wet settled volume of gelconsisting of 0.7 gm fibronectin/gm of CNBr-activated Sepharose 4B] plus1 ml of coupling buffer containing 50 μM of sodium aurothiomalate wereincubated with roller mixing for 4 hours at 37° C. The Sepharose wasthen washed with a suitable solution, for example 50 mM Tris-HCl, pH7.5, to remove unreacted gold compound and protein. A moist pellet ofgel was recovered either by centrifugation or by vacuum filtrationthrough a glass sinter and incubated with trypsin at an enzyme toprotein substrate ratio of approx 1:200 in 50 mM Tris-HCl, pH 7.5 for15-120 mins at 37° C. Released gold-fibronectin fragment conjugate wasseparated from the Sepharose by centrifugation.

Albumin-Sepharose was substituted for fibronectin-Sepharose in the aboveprocedure to obtain gold-albumin fragment conjugates.

Gold conjugates obtainable by the above procedures are suitable ascarrier protein-gold conjugates for preparation of anti-rheumaticconjugates by cross-linking to gelatin-targetting adhesive proteinfragments.

Conjugation of gold was also tested over a range of conditions(temperature, incubation time, ionic strength). The gold component ofsodium aurothiomalate was followed specifically by atomic absorptionspectroscopy and protein was measured by the Folin-Lowry assay. Table 10below shows how the amount of bound gold varied. Binding (in μ moles ofgold per unit volume of fibronectin-Sepharose) was significant but low(6-8 μM) in low ionic strength Tris-HCl. Addition of 1 M urea increasedbinding by between 50%-150% with the optimum at 4 hrs and 37° C. Table11 below shows the relative effect of increasing levels of urea onbinding gold to fibronectin-Sepharose. Increasing urea concentrationbetween 0.1 and 1.0 M increased the gold incorporation by almost fourfold (in this case gold binding was measured relative to the proteincontent of the conjugate). In fact, these are very high levels ofbinding of the order of 1.2×10⁴ moles gold/mol fibronectin. Whenalbumin-Sepharose was substituted for fibronectin-Sepharose, goldbinding was approximately 35%, on a protein weight basis, of the minimumlevel for fibronectin-Sepharose. Although coupling was at low ionicstrength, the final conjugate was largely stable to physiological NaCllevels. Loss of gold from the immobilised complex was constant at 21% to23% between 0.1 M and 0.75 M NaCl.

                  TABLE 10                                                        ______________________________________                                        Effect of reaction conditions on gold coupling                                to solid phase fibronectin                                                                  0.15 mM NaCl                                                                  50 mM Tris 1M Urea                                                      50 mM Tris      % of           % of                                           μM Au.sup.(1)                                                                      μM Au                                                                              Basal.sup.(2)                                                                          μM Au                                                                            Basal                                  ______________________________________                                        2 hr reaction                                                                 +4° C.                                                                           6.00      4.65    77.5%  9.15  152.5%                               +37° C.                                                                          7.20      3.60    50%    12.75 177%                                 4 hr reaction                                                                 +4° C.                                                                           6.75      6.30    93%    12.00 178%                                 +37° C.                                                                          7.80      6.30    81%    19.50 250%                                 ______________________________________                                    

(1) Figures refer to total Sepharose-bound gold (by atomic absorption)recovered from 2 ml of gel.

(2) Basal binding is taken as value in Tris buffer only, usingcorresponding conditions and taken as 100%

                  TABLE 11                                                        ______________________________________                                        Influence of urea concentration on conjugation of gold                        to fibronectin-Sepharose                                                      Urea      Mean Gold incorporation                                                                        % increase over                                    Concentration                                                                           (μM/μg protein)*                                                                         control level                                      ______________________________________                                        0         0.05             --                                                 0.1M      0.098             96%                                               0.5M      0.133            165%                                               1M        0.20             390%                                               2M        0.193            287%                                               ______________________________________                                         *Mean of 2 experiments; incubation of 4 hrs at 37 C.                     

A2. Direct covalent coupling of gold to fibronectin or fibronectinfragments immobilised on fibronectin-Sepharose

Fibronectin-Sepharose binds large quantities of soluble fibronectin atlow ionic strength which can be recovered by raising the buffer NaClconcentration.

1 ml of fibronectin-Sepharose slurry [1-2 parts buffer (50 mM Tris-HCl,pH7.5) plus 1 part wet settled volume of gel consisting of 0.7mgfibronectin/gm of CNBr-activated Sepharose 4B] was mixed with 2 ml ofpurified plasma fibronectin (approx. 1 mg/ml) in the same buffer andmixed for 15-30 mins. The gel was washed (same buffer) to remove unboundfibronectin and incubated with roller mixing for 4 hrs at 37° C. in 50mM Tris-HCl pH7.5 containing 1 M urea and 25 u mole of gold as sodiumaurothiomalate per mg of fibronectin loaded. The gel was then againwashed and resuspended in high ionic strength buffer (50 mM Tris-HClpH7.5: 0.5 M NaCl) to dissociate the adsorbed fibronectin-goldconjugate. Mixing was carried out at room temperature for 15-60 mins.The gold-fibronectin conjugate in solution was de-salted by dialysis,ultrafiltration or gel filtration or a combination of such techniquesand stored frozen or dry.

Using this technique for gold conjugation, 37% of the gold applied wasconjugated to give a specific binding value of 9.8×10³ moles gold/moleof fibronectin. After cross-linking to gelatin-targetting thrombinfragments of fibronectin (see section C), the specific gold binding wasreduced to 1.1×10³ mole gold/mole of fibronectin.

In the above procedure, soluble fibronectin may be replaced byfibronectin fragments to also provide carrier protein-gold conjugatessuitable for preparation of anti-rheumatic conjugates by cross-linkingto gelatin targetting adhesive protein fragments.

A3. Direct covalent coupling of gold to fibronectin in the presence ofgelatin

Using the gold conjugation methods described in A1 and A2 above, theresulting gold-fibronectin or gold-fibronectin fragment conjugates donot retain gelatin-binding activity. In this third method used fordirect coupling of gold to fibronectin or fibronectin fragments, thegelatin-binding sites of the protein employed in the coupling reactionwere protected by binding to gelatin either in the form of solublegelatin or gelatin-Sepharose.

2 mls of a 1 to 5 mg/ml solution of gelatin (prepared by denaturation ofpurified skin collagen) in 50 mM Tris-HCl pH7.5 was mixed with 2 mg ofintact fibronectin or gelatin-binding fibronectin fragments in the samebuffer. The mixture was rolled for 1 hour at room temperature.Alternatively, the fibronectin or gelatin-binding fibronectin fragmentswere adsorbed on to gelatin-Sepharose in the same buffer. 50 μmoles ofsodium aurothiomalate were added and the solution orgelatin-Sepharose-fibronectin slurry made up to 1 M with urea and mixedat room temperature for 4 hours.

Where soluble gelatin was used, the gelatin-fibronectin binding wasdisrupted by adding 4M urea, dialysing into 20 mM sodium phosphatecontaining 4 M urea at pH 5.2 and mixing with 1 gm of pre-swollencarboxymethyl cellulose ion-exchanger (Whatman CM52*). Soluble gelatinwas bound to the ion-exchanger and removed by centrifugation leaving thesoluble conjugate. ≠* trade mark

Where gelatin-Sepharose was used, the fibronectin-gold conjugate wasmade on the gel and unreacted drug or protein fragment removed bywashing with the same buffer free of gold compound. The finalfibronectin-gold conjugate was recovered by washing thegelatin-Sepharose adsorbent either with 4 M urea or 1 M arginine in 50mM Tris-HCl, pH 7.5.

Using a "gelatin binding protection method" as above with wholefibronectin, it was found possible to conjugate about 50% of the gold togive fibronectin-gold conjugate with 2.3×10³ M gold/M fibronectin.

From gelatin-binding gold-fibronectin fragment conjugates thus isolated,conjugates having an fibronectin fragment with gelatin-binding affinity,but lacking fibrin-binding affinity may be selected by FFFM-Sepharose orheparin-Sepharose affinity chromatography suitable for direct use asantirheumatic conjugates. It will be appreciated that suchanti-rheumatic conjugates may also be obtained by subjectinggelatin-binding gold-whole fibronectin conjugates isolated by aprocedure as above to protease digestion, with subsequent selection ofgold-fibronectin fragment conjugates lacking fibrin-binding affinity.Alternatively gold-fibronectin conjugates, as well as gold-fibronectinfragment conjugates prepared by a "gelatin-binding protection method" asabove, may be employed as carrier protein-gold conjugates forpreparation of anti-rheumatic conjugates by cross-linking togelatin-targetting adhesive protein fragments, e.g. fibronectinfragments having gelatin-binding affinity, but substantially lackingfibrin-binding affinity.

B. Preparation of Gelatin-Binding Fragments of Fibronectin LackingFibrin-Binding Affinity

Fibronectin was digested with trypsin or thrombin as described inSections C1 and C2 of Example 1 or with cathepsin D in conventionalmanner to yield a mixture of fragments with either or bothgelatin-binding affinity and fibrin-binding affinity or neither of thesetwo binding specificites. 10 ml of a 1 mg/ml solution of proteasedigested fibronectin in 50 mM Tris-HCl, pH 7.5 was applied to aheparin-Sepharose column. Unbound material (free of fragments with aheparin or fibrin binding site) was applied to a gelatin-Sepharosecolumn at 1 mg/ml in 50 mM Tris-HCl, pH7.5 containing 0.5 M NaCl. Afterwashing off unbound protein, gelatin-binding fragments were eluted witheither 4 M urea or 1 M arginine in running buffer and desalted bydialysis.

C. Cross-linking of Carrier Protein-Gold Conjugates to GelatinTargetting Fibronectin Fragments

5 ml of fibronectin carrier-gold conjugate (prepared by a procedure asdescribed in A1, A2 or A3 above) in 10 mM sodium phosphate buffer pH 5.2was made up to a final concentration of 1% (0.24 M) cyanamide andincubated at 22° C. for 1 to 4 hours with 5 ml of gelatin-bindingfibronectin fragments prepared as in B above, at the same proteinconcentration as the fibronectin-gold conjugate. Excess reactants wereremoved by dialysis, ultra-filtration or gel filtration.

D. Testing for Gelatin Binding Activity of The Final Conjugate

The final conjugate was assayed for gelatin binding activity by applyingit to a gelatin-Sepharose column as in B above. Bound material waseluted and assayed for total protein and for gold content. This gave ameasure of gelatin-binding as μM of gold/μg gelatin-binding protein.

I claim:
 1. A pharmaceutically active conjugate comprising an anti-rheumatic agent for treating rheumatic disease, wherein said anti-rheumatic agent is conjugated directly or indirectly with at least one fragment of fibronectin having improved gelatin-binding specificity compared with that of a whole parent fibronectin molecule, wherein said fibronectin fragment does not exhibit cell attachment activity.
 2. A conjugate as claimed in claim 1 wherein the said anti-rheumatic agent is covalently conjugated.
 3. A conjugate as claimed in claim 1 wherein the said anti-rheumatic agent is selected from gold, anti-rheumatic gold compounds and penicillamine.
 4. A conjugate as claimed in claim 1 wherein the anti-rheumatic agent and said conectin fragment(s) are conjugated directly or indirectly to a carrier molecule.
 5. A conjugate as claimed in claim 4 wherein the said carrier is selected from fibronectin or albumin.
 6. A conjugate as claimed in claim 4 wherein the said carrier is covalently conjugated.
 7. A method for treating a rheumatic disease in a human or non-human animal, comprising administering to said human or non-human animal by an effective route and in an effective amount a pharmaceutically active conjugate comprising a pharmaceutically active substance for treating said rheumatic disease, wherein said pharmaceutically active substance is conjugated directly or indirectly with at least one fragment of fibronectin having improved gelatin-binding specificity compared with that of a whole parent fibronectin molecule, wherein said fibronectin fragment does not exhibit cell attachment activity.
 8. The method of claim 7, wherein said pharmaceutically active conjugate is administered to a human.
 9. The method of claim 7, wherein said pharmaceutically active substance is selected from the group consisting of gold, anti-rheumatic gold compounds and penicillamine.
 10. A method for preparing a pharmaceutically active conjugate for treating rheumatic disease, comprising the step of conjugating a pharmaceutically active substance directly or indirectly to at least one fragment of fibronectin, said fibronectin fragment having improved binding specificity for gelatin compared with that of a whole parent fibronectin molecule, wherein said fibronectin fragment does not exhibit cell attachment activity.
 11. The method of claim 10, wherein said fibronectin fragment is a component of a protease digestion mixture of said a whole parent fibronectin molecule.
 12. The method of claim 10, wherein said pharmaceutically active substance is covalently conjugated to a carrier, and said carrier is then covalently conjugated to one or more of said fibronectin.
 13. The method of claim 10, wherein said pharmaceutically active substance is not a protein.
 14. The method of claim 13 wherein said carrier is a protein containing sulphydryl groups.
 15. The method of claim 14 wherein gold is directly conjugated to said carrier protein via said sulphydryl groups by reacting said carrier protein with aurothiomalic acid or a salt thereof.
 16. The method of claim 15 wherein said carrier protein-gold conjugate is digested by a protease and a resultant protein-gold conjugate is covalently conjugated to said one or more fibronectin.
 17. A method of preparing a pharmaceutically active conjugate for treating rheumatic disease, comprising the steps of:(a) conjugating a pharmaceutically active, non-protein substance directly or indirectly to a whole parent fibronectin molecule to form a pharmaceutically active conjugate; (b) digesting said pharmaceutically active conjugate with a protease; and (c) selecting a pharmaceutically active fibronectin fragment conjugate having improved gelatin-binding specificity compared to that of said whole parent fibronectin molecule wherein said fibronectin fragment does not exhibit cell attachment activity.
 18. The method of claim 17 which further comprises protecting binding sites on the whole parent fibronectin molecule specific to said gelatin prior to proteolysis. 